Human papillomavirus (HPV) affects up to 75% of adults and is categorized as high risk (HR) or low risk (LR) based on its association with cancer. Although there are preventive vaccines against specific HPV genotypes, immunizations have had poor uptake and completion. This leaves many women and men at continued risk of HPV infection and HPV-associated cancers, some of which are increasing in incidence. Knowing how HR HPV infections affect their host cells is critical for understanding tumor virus biology and for developing screening and treatments that reduce morbidity and mortality worldwide. Studies in HPV- associated cancers have focused on the malignant potential of HR E6 and E7 viral oncoproteins and their roles in gene activation and protein degradation; however, less well-studied is their role in post-transcriptional cellular gene regulation. We found HR HPV 16E6 (16E6), through its partnership with the endogenous protein NFX1-123, post-transcriptionally upregulated expression of the catalytic subunit of telomerase (hTERT) and a master regulator of growth and differentiation (Notch1). Little is known about the normal function of NFX1-123 in epithelial cells; however, based on the protein domain motifs and partnerships, and cellular localization of NFX1-123, we theorize NFX1-123 participates in RNA binding and regulation in healthy epithelial cells. Indeed, NFX1-123 is needed to increase Notch1 and hTERT mRNA expression by 16E6, and these increases require the NFX1-123 RNA processing protein motifs. This project seeks to identify the global set of mRNAs directly bound by NFX1-123, with and without 16E6, using a novel technique, Photoactivatable-Ribonucleoside- Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP), with the depth and breadth of Next Generation Sequencing, and the ease of nanoString Technologies. In Aim 1, we will identify mRNAs directly bound by NFX1-123 in healthy epithelial cells. We hypothesize multiple mRNAs are bound by NFX1-123 as a part of their typical processing and translation, and we have found mRNAs increased with overexpressed NFX1-123 in a large microarray. We will use PAR-CLIP as an unbiased approach to identify novel RNAs bound by NFX1- 123 and to determine whether mRNAs increased in our microarray were due mechanistically to greater amounts of RNA-protein binding by NFX1-123. In Aim 2, we will identify mRNAs bound by NFX1-123 in epithelial cells expressing 16E6, or the full HPV 16 genome, as a model for infected epithelial cells. These two sets of mRNAs bound by NFX1-123 may be mutually exclusive, as HR HPV targets new mRNAs to be bound and upregulated by NFX1-123, or they may overlap in part by HR HPV modulating an mRNA or a pathway that NFX1-123 already typically regulates. The proposed work will use cutting-edge techniques to yield a comprehensive view of post-transcriptional regulation by NFX1-123 and a HR E6 viral oncogene. These studies will lead to new information on mechanisms and pathways critical, and even treatable, in HPV infections and associated cancers.